Cholesterol is a rigid, hydrophobic molecule that plays essential roles in membrane function and in the synthesis of bile acids and steroid hormones. Cholesterol also plays a central role in the pathogenesis of two major human diseases, coronary heart disease and gallstones. Accordingly, the metabolic pathways that maintain cholesterol homeostasis have been extensively studied. In 2000, the Hobbs-Cohen laboratory showed that cholesterol excretion is mediated by a heterodimeric ATP-Binding Cassette (ABC) transporter comprising two hemi-transporters ABCG5 (G5) and ABCG8 (G8). Mutations that disrupt the function of the transporter profoundly alter cholesterol metabolism and cause severe premature coronary heart disease. Subsequent studies showed that G5 and G8 heterodimerize in the endoplasmic reticulum and traffic to the apical surfaces of enterocytes and hepatocytes, where they transport cholesterol and other neutral sterols into bile. Genetic ablation of G5G8 essentially abolishes biliary excretion of neutral sterols, and biochemical studies using purified G5G8 protein confirmed that cholesterol is the direct substrate of the transporter. The overall goal of this Project is to define the biological role and functional mechanism of G5G8. Funding from this grant has supported a sustained research effort that has elucidated the role of G5G8 in normal physiology and the clinical consequences and pathogenic mechanism of both loss and gain of transporter function. This application will focus on the functional mechanism of G5G8 to address a major unresolved question regarding G5G8 function: How does G5G8 use the energy of ATP hydrolysis to translocate a rigid, hydrophobic, neutral sterol molecule across biological membranes? It is generally accepted that ABC transporters use ATP to power changes in conformation that result in translocation of substrate across the membrane. As a first step towards defining the structural basis of sterol transport by G5G8, X-ray crystallography was used to determine the structure of G5G8 in an inward-facing (IF), open conformation, which precedes binding of ATP to the transporter and allows for the binding of substrate. To define the conformational changes that drive sterol efflux the structures of G5G8 in the outward facing conformation (OF), in which substrate is moved to the exterior face of the cell, will now be determined. In Specific Aim 1, state-of-the-art technologies (both X-ray crystallography and cryo-EM) will be used to obtain high resolution structures of G5G8 in different states of the transport cycle. The structures will be used to generate and test models of how ATP hydrolysis leads to substrate transport. Specific Aim 2 will examine the sterol binding site and the trajectory along which sterols are translocated across the membrane. To accomplish these aims, we have assembled a multidisciplinary team of investigators with expertise in physiology, biochemistry, synthetic chemistry and structural biology.